Microarray-based Expression Profiles Reveal Molecular Characterization Of Breast Duct And Lobule

Objective:This study aims to extract RNA from tissues and to verify the different geneexpressions between breast duct and lobule structure by using microarray. To discuss therelationship between these different gene expressions and the development of breastcancer and provide basic data for gene research of breast cancer.Materials and methods1. Clinical specimen collection: During2011Jan to2011Jun,select8benign breastcancer patients with nipple discharge required surgical treatment in Guangdongwomen and children’s hospital, and then conduct segmented resection and strip out themain ducts along with blue blot after injecting0.2ml of methylene blue dye intodischarge hole. lobule are collected by incise non-blue-stained glandular tissue at theterminal of specimens with volume about the size of soybean, both of them arepreserved with RNA later reagent and confirmed by separate a little part forpathological examination concurrently2. Whole gene expression detection: To elucidate gene expression differences betweenDuct and小叶, we analyzed8pairs of breast duct and lobule tissue samples from8patients with benign breast tumor using agilent gene expression microarrays. Thehybridized slide was washed and scanned using an Agilent Microarray Scanner. Rawdata of obtained images were extracted using Agilent Feature Extraction Software.Extracted data were analyzed using Agilent gene spring GX11.5. Genes wereconsidered robustly expressed and retained for further analysis if the probe intensitywas not below than400after global mean normalization in at least8samples.Differentially expressed genes were assessed using SAM (Significance analysis of microarrays) software. Significance threshold was fold changes greater than2.0and qvalue less than0.05.The significant enrichment of Gene Ontology (GO) terms of thedifferentially expressed genes was further analyzed using hyper-geometric distributionin the R language package software.3. Validation of the differentially expressed genes using quantitative RT-PCR: Tofurther validate the microarray results, we randomly chose17genes with significantlydifferential expression between the duct and lobule samples identified by microarrayfor further validation using quantitative RT-PCR. The selected genes include ADIPOQ,ADORA2B, BMP7, CA4, CCL19, ITGB3, CCL21, CEL, ECSCR, FABP4, LOX,S100A8, HSD11B2, LAMA1, LILRB3, S100A9and WNT5A. The ratios of averageexpression levels of each gene between the duct and lobule tissues were measuredusing the same batch of samples as microarray.Results1. Differentially expressed genes between breast duct and lobule showed by geneexpression microarray Using Agilent human8*60K microarray platform, theexpression profiles of breast duct and lobule samples were analyzed. Differentiallyexpressed genes were assessed using SAM (two class unpaired), with the significancethreshold of fold changes greater than2.0and q value less than0.05. A total of426transcripts were identified as being significantly differentially expressed (A full list ofdifferentially expressed genes is presented in the supplementary material). Supervisedhierarchical clustering based on the426differentially expressed genes showed distinctclustering of the duct and lobule samples.The significant enrichment analysis of GOterms for the differentially expressed genes using the R language package softwaredemonstrated that these426differentially expressed genes are involved in manyimportant biological processes, including negative regulation of low-densitylipoprotein receptor biosynthetic process, response to steroid hormone stimulus andoxidation reduction.2. Validation of the differentially expressed genes using qRT-PCR assay To furthervalidate the microarray results, we randomly chose17genes with significantlydifferential expression between the duct and lobule samples identified by microarrayfor further validation using quantitative RT-PCR. The selected genes include ADIPOQ, ADORA2B, BMP7, CA4, CCL19, ITGB3, CCL21, CEL, ECSCR, FABP4, LOX,S100A8, HSD11B2, LAMA1, LILRB3, S100A9and WNT5A. The ratios of averageexpression levels of each gene between the duct and lobule tissues were measuredusing the same batch of samples as microarray. Fold changes measured by microarraywere closely correlated those measured by quantitative RT-PCR, confirming largedifferences in gene expression between duct and lobule.